Trafficking of macromolecules between the nucleus and cytoplasm is mediated by nucleocytoplasmic shuttling receptors, which translocate cargo through the nuclear pore complex (NPC) by binding to a series of nucleoporins. The small GTPase Ran plays a critical role in nuclear import by modulating the interaction of cargo with these transport receptors. Several groups have shown that in vitro nuclear import of certain small cargoes by the importin alpha/importin beta and transportin receptor pathways can occur in the absence of Ran and/or NTP hydrolysis. By contrast, recent work from the Gerace lab has shown that both Ran and hydrolyzable GTP are needed for the efficient import of certain large cargoes by these pathways. To accommodate these findings, a model is proposed where the requirements for Ran and energy in nuclear import of a cargo-receptor complex is governed by the diffusion rate of the complex and by the affinity of the complex for nucleoporins. The aims of this proposal are directed at testing this model in detail by examining the effects of cargo size and affinity of the transport complex for nucleoporins in the importin alpha/importin beta, snurportin/importin beta, and the transportin receptor pathways. These studies are expected to provide insight on the basic mechanism of translocation through the NPC and the Ran requirement in this process. This is a fundamental process that is important for the biology of the cell in a wide range of normal conditions and may be altered in certain pathological states.